Hydrogen sulfide (H2S) is produced in the brain, liver and in the wall of many blood vessels. There is increasing evidence that H2S is also produced in the gastrointestinal tract and that it serves important physiological functions. Our overall hypothesis is that endogenously generated H2S in the muscle wall is a physiological signaling gas in the colon and that endogenously generated H2S in extrinsic sympathetic prevertebral ganglia modulates the excitability of ganglion neurons that innervate the colon. We hypothesize that under normal conditions, H2S is enzymatically produced and released in the muscle wall of the colon and prevertebral ganglia and that it regulates the excitability of circular smooth muscle cells and the excitability and synaptic transmission of myenteric ganglion neurons and prevertebral ganglion neurons. These hypotheses will be tested by a number of diverse experiments and novel approaches. Our work is organized under 5 Specific Aims. We will determine the cellular distribution of the major synthesizing enzymes for H2S, the endogenous capacity of intact tissue to generate H2S, the effect of H2S on smooth muscle membrane potential and the physiological interplay between H2S and carbon monoxide, the cellular and molecular mechanisms(s) that mediate the hyperpolarizing response to H2S in circular smooth muscle cells and the physiological effect of H2S on excitability and synaptic transmission in myenteric and prevertebral ganglion neurons. Our methods will include immunohistochemistry, confocal microscopy and 3D volume reconstruction methodology, gas chromatography to measure H2S production, microelectrode recordings from smooth muscle cells, myenteric and prevertebral ganglion neurons, patch clamp recordings to measure membrane potential and currents of dissociated smooth muscle cells, RT PCR and RT-qPCR. We anticipate making significant progress toward establishing that H2S is an important physiological regulating molecule in the gastrointestinal tract and translate this knowledge into a better understanding of the potential role H2S might have in disordered motility in humans. PUBLIC HEALTH RELEVANCE: In the work outlined in this proposal, we will focus on the role of endogenous hydrogen sulfide (H2S) in thephysiology of colonic smooth muscle, colonic enteric ganglion neurons and sympathetic ganglion neurons thatinnervate the colon. This work will provide the first quantitative information on the release of endogenous H2Sfrom any intact and living tissue as well as the regulation of H2S release, the first examination of the role of H2Sin modulating colonic electrical and contractile activity in experimental animal models and in the human and the role of H2S in ganglion neurons that regulate colonic motor activity. We anticipate our studies will provide mechanistic information relevant not only to the gastrointestinal tract but also to other organs in the body that generate H2S such as the liver, vasculature and brain.